Abstract

Several naturally occurring dietary polyphenols with chemopreventive or anticancer properties are topoisomerase II poisons. To identify additional phytochemicals that enhance topoisomerase II-mediated DNA cleavage, a library of 341 Mediterranean plant extracts was screened for activity against human topoisomerase IIα. An extract from Phillyrea latifolia L., a member of the olive tree family, displayed high activity against the human enzyme. On the basis of previous metabolomics studies, we identified several polyphenols (hydroxytyrosol, oleuropein, verbascoside, tyrosol, and caffeic acid) as potential candidates for topoisomerase II poisons. Of these, hydroxytyrosol, oleuropein, and verbascoside enhanced topoisomerase II-mediated DNA cleavage. The potency of these olive metabolites increased 10-100-fold in the presence of an oxidant. Hydroxytyrosol, oleuropein, and verbascoside displayed hallmark characteristics of covalent topoisomerase II poisons. (1) The activity of the metabolites was abrogated by a reducing agent. (2) Compounds inhibited topoisomerase II activity when they were incubated with the enzyme prior to the addition of DNA. (3) Compounds were unable to poison a topoisomerase IIα construct that lacked the N-terminal domain. Because hydroxytyrosol, oleuropein, and verbascoside are broadly distributed across the olive family, extracts from the leaves, bark, and fruit of 11 olive tree species were tested for activity against human topoisomerase IIα. Several of the extracts enhanced enzyme-mediated DNA cleavage. Finally, a commercial olive leaf supplement and extra virgin olive oils pressed from a variety of Olea europea subspecies enhanced DNA cleavage mediated by topoisomerase IIα. Thus, olive metabolites appear to act as topoisomerase II poisons in complex formulations intended for human dietary consumption.

Structures of olive plant metabolites. Polyphenols present in P. latifolia L. and other olive species, including hydroxytyrosol, oleuropein, verbascoside, caffeic acid, and tyrosol, are shown. Hydroxytyrosol (red) is a component of oleuropein and verbascoside, and caffeic acid (blue) is a component of verbascoside.

Activity of olive metabolites against topoisomerase IIα and IIβ is enhanced by the presence of an oxidant. (A) The effects of an oxidant, K3Fe(CN)6, on DNA cleavage mediated by topoisomerase IIα were determined in the presence of 100 μM olive metabolites [hydroxytyrosol (HT; red), oleuropein (OE; green), verbascoside (VERB; purple), caffeic acid (CA; blue), and tyrosol (TY; black)] or in the absence of a metabolite (TII; gray) (left). The effects of olive leaf metabolites on DNA cleavage were determined in the presence of 10 μM K3Fe(CN)6 (right). (B) The effects of olive metabolites on DNA cleavage mediated by human topoisomerase IIβ were determined in the presence of 10 μM K3Fe(CN)6. DNA cleavage levels were calculated relative to a control reaction mixture that contained no metabolite and no oxidant. Error bars represent standard deviations for three independent experiments.

Effects of olive leaf metabolites on the sites of DNA cleavage generated by topoisomerase IIα. An autoradiogram of a polyacrylamide gel is shown. Reaction mixtures contained no enzyme (DNA), enzyme in the absence of metabolite (TII), or enzyme in the presence of 10 μM hydroxytyrosol (HT), oleuropein (OE), or verbascoside (VERB) in the presence of 10 μM K3Fe(CN)6. A control DNA cleavage reaction mixture that contained 20 μM etoposide also is shown. The autoradiogram is representative of three independent experiments.

Effects of olive leaf metabolites on DNA cleavage complex stability. (A) The ability of human topoisomerase IIα to ligate cleaved DNA is shown. Reactions (20 s) were conducted in the presence of no metabolite (TII; gray), hydroxytyrosol (HT; red), oleuropein (OE; green), verbascoside (VERB; purple), or 100 μM etoposide (ETOP; black). Reaction mixtures contained 1 mM metabolite and no oxidant (left) or 10 μM metabolite in the presence of 10 μM K3Fe(CN)6 (right). (B) The effects of olive metabolites on the persistence of topoisomerase IIα–DNA cleavage complexes in the absence or presence of oxidant are shown. Assays were conducted in the presence of 1 mM metabolite (empty circles) or 10 μM metabolite with 10 μM K3Fe(CN)6 (filled circles). Colors are as described above. For the ligation and persistence reactions, DNA cleavage levels at time zero were set to 100% to allow a direct comparison. Error bars represent the standard deviation of at least three independent experiments.

Olive metabolites are covalent topoisomerase II poisons. (A) Effects of DTT on the ability of olive metabolites to enhance DNA cleavage mediated by topoisomerase IIα. DNA cleavage reactions were performed in the absence of DTT (filled bars, No DTT), in the presence of 100 μM DTT that was added after the cleavage–ligation equilibrium was established (stippled bars, Post DTT), or in the presence of 100 μM DTT that was added at the start of the reaction (empty bars, Pre DTT). Reaction mixtures contained 1 mM hydroxytyrosol (HT; red), oleuropein (OE; green), or verbascoside (VERB; purple). DNA cleavage levels were calculated relative to a control reaction mixture that contained no metabolite. (B) Effects of 3,4-dimethoxyphenylethanol and 4-hydroxy-3-methoxyphenylethanol on topoisomerase IIα-mediated DNA cleavage. The effects of 500 μM 3,4-dimethoxyphenylethanol (black bar) or 4-hydroxy-3-methoxyphenylethanol (white bar) on the cleavage of negatively supercoiled plasmid DNA by topoisomerase IIα were determined in the presence 10 μM K3Fe(CN)6. Data for reaction mixtures that contained no compounds are colored gray. DNA cleavage levels were calculated relative to a control reaction mixture that contained no compounds or oxidant. In all cases, error bars represent standard deviations for three independent experiments.

Olive metabolites inhibit topoisomerase IIα when incubated with the enzyme prior to DNA. The effects of hydroxytyrosol (HT; red), oleuropein (OE; green), and verbascoside (VERB; purple) are shown. Metabolites were incubated with the human enzyme in the absence of oxidant (1 mM metabolite, filled circles, left) or in the presence of 10 μM K3Fe(CN)6 (10 μM metabolite, empty circles, right). DNA cleavage levels were calculated relative to a control reaction mixture to which the metabolite was added after the addition of DNA to assay mixtures. Error bars represent standard deviations of at least three independent experiments.

Olive metabolites require the N-terminal domain to enhance DNA cleavage mediated by topoisomerase IIα. The effects of olive metabolites on DNA cleavage mediated by topoisomerase IIα lacking the C-terminal domain (Δ1175) or both the C-terminal and N-terminal domains (Catalytic core) are shown in panels A and B, respectively. DNA cleavage reactions were performed using 1 mM metabolite [hydroxytyrosol (HT; red), oleuropein (OE; green), or verbascoside (VERB; purple)] in the absence of an oxidant (filled bars) or 10 μM metabolite in the presence of 10 μM K3Fe(CN)6. Results with no metabolite (TII, gray) or 100 μM etoposide (ETOP, black) in the absence or presence of an oxidant are shown as controls. DNA cleavage levels were calculated relative to scission generated by restriction endonucease EcoRI, which was set to 100%. Error bars represent the standard deviation of at least three independent experiments. Baseline levels of DNA cleavage generated by the catalytic core are lower than those generated by full-length topoisomerase IIα.